Caffeic Acid and C. elegans’ Protection Against Toxin Induced NeurodegenerationBy: Michelle Chyn
Degree of PD in Patients DM US $ pounds
Average person 3,920 2,590 1,690
Fluctuations in performance 6,010 3,970 2,580
More stable condition 2,700 1,780 1,160
Hoehn and Yahr scale stage I 2,230 1,470 9,60
Hoehn and Yahr scale stage V 11,870 7,830 5,100
Need Parkinson’s Disease
(PD)- 500,000 people in U.S.
$6,000,000,000 spent annually for PD treatment
National Institute of Neurological Disorders and Stroke
Dodel RC, Pepperl S, Köhne-Volland R, Szucs T, Werhahn KJ, Noachtar S, Oertel WH. “Costs of drug treatment of neurologic diseases: Parkinson disease, dystonia, epilepsy.” European Journal of Neurology. Vol. 7. Pp. 479-85 1991.
Figure 1: Cost of PD treatment per person annually
http://www.parkinsonsdecisionaid.eu.com/images/ac/2008/incidence.gif
http://www.e-importz.com/images/coffee_consumption.gif
Coffee consumption in North America and Europe = 1/3 of the tap water intakeNeed Cont.
Figure 2: PD in European Countries
Figure 3: Coffee Consumption
Knowledge Base
C. elegans biosynthesize dopamine in the corpus stratum (basil ganglia) (Braugart, 2004)
Figure 4:
http://en.wikipedia.org/wiki/Basal_gangliaFigure 5: 3-hydroxytyramine (Dopamine)
http://pubchem.ncbi.nlm.nih.gov/summary/summary.cgi?cid=681&loc=ec_rcs
Knowledge Base Cont.
Copper is a neurotoxin when consumed in excess amounts (Gaggelli et al., 2006)
Figure 6: Copper oxidehttp://en.wikipedia.org/wiki/Copper(I)_oxide
Literature Review Antioxidants in coffee decreases risk of development
of age-related diseases (Eskelinen et. al, 2009) Caffeine intake doesn’t show health benefits (Lopez-
Garcia, et. al, 2008)
http://pubchem.ncbi.nlm.nih.gov/summary/summary.cgi?cid=689043&loc=ec_rcs
Figure 7: Caffeic acid
Literature Review Cont. Caenorhabditis elegans MPP+ model used as
PD model for testing anti-PD drugs (Braungart, et al. 2004)
Figure 8: Dose-response effect of MPP+ treatmenton the mobility of wild-type C. elegansanimals.
http://content.karger.com/ProdukteDB/produkte.asp?Aktion=ShowPDF&ArtikelNr=80983&Ausgabe=230458&ProduktNr=229093&filename=80983.pdf
Literature Review Cont. Green Florescent Protein (GFP) florescence
shown in dopaminergic neurons of transgenic strains of C. elegans (Braungart, et al., 2004)
http://content.karger.com/ProdukteDB/produkte.asp?Aktion=ShowPDF&ArtikelNr=80983&Ausgabe=230458&ProduktNr=229093&filename=80983.pdf
Figure 9: GFP expression in dopaminergic neurons of untreated and MPP+ treated worms
Literature Review Cont. Direct addition of dopamine-2-c did not help
increase noradrenaline presence in PD patients (Goodall and Alton, 1969)
http://www.pubmedcentral.nih.gov/picrender.fcgi?artid=297487&blobtype=pdf
Figure 10: % noradrenaline recovery after dopamine-2-c infusion
Purpose Observing the effect of caffeic acid on
neurotoxin-induced degeneration in C. elegans
H0- caffeic acid will not affect the degeneration rate in the C. elegans
HA- caffeic acid will decrease degeneration in the C. elegans.
Hypothesis
MethodologyCompounds in Coffee and C. elegans Protection Against Parkinson’s Disease
Caenorhabditis elegans egIs1[Pdat-1::GFP] strain from Caenorhabditis Genetics Center (CGC)
N=90
C. elegans cultured at 20°C in NGM plates in normal oxygen environment. Caffeic acid and MMP+ concentrations inserted in food of E. coli for 3 days at
L1 stage. Organisms then moved out into normal NGM plates.
Control
addition of copper oxide, concentratin according to LD 50
N=30
Group 2
addition of copper oxide
caffeic acid- lower concentration according to LD 50
N=30
Group 3
addition of copper oxide
caffeic acid- higher concentration according to LD 50
N=30
Statistical analysis using SPSS and ANOVA
Data collected through stereo florescent microscope viewing of GFP florescence on a scale of normal, weak, and none, and area through
Adobe Photoshop on the 3rd day.
Protocols
Eggs laid overnight at L4 stage on fresh NGM plates (Mehta, et al, 2009)
Picture drawn by Victoria Wei
Figure 11: Culturing of C. elegans on a petri dish
Protocols Cont.
http://www.scq.ubc.ca/wp-content/uploads/2006/07/wormcycle.gif
N=30 per group, total N=90
Group 1: Copper oxide
Group 2: Copper oxide and Caffeic acid
Group 3: Copper oxide and Caffeic acid
Figure 12: C. elegans growth stages
Protocols Cont. Data collect on scaled brightness of GFP
expression (Braungart, et al., 2004) and area of florescence on Photoshop
Data analysis using SPSS
http://pubchem.ncbi.nlm.nih.gov/summary/summary.cgi?cid=33557&loc=ec_rcs
Figure 13: Sodium azide (NaN3)
Figure 14: Fluorescent Microscope
http://www.niams.nih.gov/Research/Ongoing_Research/images/ost_images/leica_dmr_fluor_micro.jpg
Protocols
Do-Ability Equipment available:
GFP filter and florescent microscope UV lamp Petri dishes Photoshop
Equipment needed: C. elegans pdat-1::GFP strain from CGC E. coli OP50 strain from CGC Copper oxide, sodium azide, and caffeic acid
from Sigma NGM from Carolina Biological
BudgetVendor Cat # Pg. Item Qty. Description Unit $ Total $
Caenorhabditis Genetics Center BZ555
C. elegans pdat-1::gfp 110 $7.00 $7.00
Sigma C0625 Caffeic Acid 1 5 g $53.30 $53.30
Carolina Biological 173520
Nematode Growth Agar 2 135 mL $6.25 $12.50
Caenorhabditis Genetics Center E. coli OP50 $7.00 $7.00
Carolina Biological 741270 petri dishes 10100 x 15 mm $6.45 $64.50
Sigma S2002 Sodium azide 1 25 g $21.20 $21.20
Carolina Biological 216880 ampicillin 1 5 g $43.25 $43.25
Sigma F0503 fluorodeoxyurodine 1 100 mg $117.00 $117.00
Sigma 208825 copper 1 25 g $28.30 $28.30
Total Cost $354.05
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